SIU School of Medicine

Jump directly to a section:

Pharmacology

Pharmacology Imaging Facility 

If Interested in use of equiptment please contact Donald Caspary, Ph.D, at dcaspary@siumed.edu

Description of Instrument

Instrument:MBF Bioscience imaging system
Supplier:MBF Bioscience

Description: The requested MBF Bioscience imaging system includes two software programs: Neurolucida and Stereo Investigator.  Neurolucida (http://www.mbfbioscience.com/neurolucida) is a program that permits neuronal tracing, 3D mapping, high-resolution imaging analysis and morphometry. The imaging data can be acquired at multiple Z-axis levels capturing the entire 3D extent of neurons and brain regions. This capability is essential in research projects where neuronal morphology is expected to correlate with a specific neuronal function, or responsiveness to a given drug, or expression of a given protein. The program provides an automated analysis of numerous quantitative parameters (e.g., branch length, diameter, Sholl analysis, etc.) and converts the results into standard document formats ready for presentation or publication. Stereo Investigator (http://www.mbfbioscience.com/stereo-investigator/stereo-investigator) is a program for unbiased stereological analysis permitting a comprehensive estimation of cell populations and morphometric properties of biological structures including quantification of cell counts, length, areas and volumes. This software performs unbiased systematic random sampling through incorporation of automated stage movement, sampling site placement and user-friendly graphic displays for rapid analysis of tissue samples.
Both software programs provide powerful tools in acquisition of images from diverse image modalities such as brightfield, fluorescent, confocal and MRI. The data can be acquired from live image digital or video cameras, stored image files from brightfield, confocal or electron microscopes. The computer software programs control a motorized XYZ stage from integrated navigation through tissue sections permitting an advanced analysis from different view points. The image sections are then integrated into a single data file for a comprehensive 3D quantitative morphometric analysis which includes 3D measurements, reconstruction of branching processing and delineation and mapping anatomical regions. Moreover, additional Plug-In modules are available for confocal and MRI analysis, 3D modeling and virtual slide creation.
The main advantage of MBF system is that unlike its competitors, MBF system offers a complete set of data acquisition and analysis tools all in one package (see below for comparison with competitors).

Hardware. The Zeiss AxioImager M1 microscope will be equipped with the confocal Apotome apparatus and a color digital camera for brightfield microscopy with a removable IR filter for fluorescent microscopy (Microfire, Optronics, Goleta, CA), 10 position motorized filter wheel, high resolution position encoded motorized stage (0.05μm step resolution 0.1μm accuracy; Ludl, Hawthorne, NY) and motorized focus control with linear encoder (0.05μm step; Ludl). The system will include standard MBF functionality, such as lens calibration, camera alignment and motorized stage alignment, and filter wheel designation. An isolation platform will provide positional stability during imaging (Vistek, Phoenix, AZ). Software control from Stereo Investigator and Neurolucida allow switching of filters, shutter control, microscope stage XYZ-movement and camera control.

Stereo Investigator: Systematic random sampling done by the integrated Stereo Investigator program allows rapid and concise image gathering across the tissue which obviates the need to exhaustively sample the tissue section prior to applying stereological probes such as optical fractionator, space balls, nearest neighbor and other probes used in neuroscience research. The WorkFlow goes through the step-by-step procedure of an optical fractionator - from configuring the study through analyzing data. Visible indicators at each step requiring attention provide alerts and other relevant options. The WorkFlow encapsulates all the necessary commands to make the optical fractionator simple to perform which drastically reduces the software training required by other systems. The WorkFlow also manages the software control settings to seamlessly move between tasks by removing redundant manual adjustments. Image stacks collected within Stereo Investigator are maintained in registry with other image and data files, and can be transferred to workstation computers to allow efficient use of the Zeiss M1 microscope for data collection not analysis.
Neurolucida: Neurolucida is advanced scientific software for performing brain mapping, neuron tracing, anatomical mapping, and morphometry. Neurolucida’s Serial Section Manager integrates unlimited sections into a single data file, maintaining each section in appropriate 3D space for full quantitative analysis. Neurolucida’s neuron tracing capabilities include 3D measurement and reconstruction of branching processes. In addition, sophisticated tools exist for mapping anatomical regions for detailed morphometric analyses. As described in the Project Description of the grant, Neurolucida will be used to map and quantify neuronal morphology. Like Stereo Investigator, all image files have an embedded coordinate system to allow registration between other related image stacks (to make montages, for example) or data files. All image files can be analyzed off-line by transfer to workstation versions of the software on a desktop PC.
Virtual Slide: The Virtual Slice Module is a plug-in extension for the Neurolucida and Stereo Investigator software that provides the additional capability of creating extremely high-resolution montages composed of images obtained from multiple microscopic fields-of-view. The resolution of the individual fields is maintained in the montage, allowing for the capacity to zoom in and out without loss of resolution. The Virtual Slice Module uses a motorized stage to automatically collect a series of contiguous images of a specimen and merges them into a single image montage. Automatic correction is done to obtain images with even illumination. The resulting montage is virtually seamless and can easily exceed the resolution and the field size of photomicrographic images. A particular benefit of Virtual Slice will be that each analysis, whether neuron tracing or evaluations of neuron numbers etc., is associated with the image file. This feature allows researchers to share their data as well as the full image context from which it was obtained.

AutoNeuron: AutoNeuron, available as a plug-in module for Neurolucida, reconstructs neurons in 2D or 3D rapidly in either fully automatic or interactive mode. The AutoNeuron tracing time for a typical confocal or brightfield stack is under one minute on a standard desktop PC, saving hours of manual tracing time compared to the current computer assisted approach. Available as a plug-in module for Neurolucida, the reconstruction model created by AutoNeuron can be viewed and edited within Neurolucida. AutoNeuron models neuronal trees as branching structures, complete with branch nodes, roots and endings.

Comparison of Neurolucida with competitors:
            Neurolucida is by far the most popular choice for a computer-controlled system for cellular tracing and reconstruction in three dimensions. The only two software programs that offer somewhat analogous tools are: Filament Tracer (Bitplane Software) which offers solely automated reconstruction; and 2) ImageJ (opensource) which does not offer a complete and ready to use solutions for imaging analysis; does not offer software and hardware support and often requires considerable programming for a desired imaging analysis. More importantly, unlike its competitors, MBF Bioscience offers a complete set of data acquisition and analysis tools all in one package. For example, although Filament Tracer (Bitplane) offers automated tracing, it does not control microscopic hardware and requires the automated reconstruction from fluorescent image stacks only (i.e., no reconstruction from brightfield image stacks). Unlike AutoNeuron (a Neurolucida module), Filament Tracer does not allow manual tracing and analysis (i.e., the option for editing of the trace once it is completed). While ImagePro (Media Cybernetics) and Volocity (Improvision) offer image analysis, they do not offer 3D neuron reconstruction capabilities. By contrast, Neurolucida permits reconstruction of cells and regions that extend across multiple fields of view at the desired magnification required to visualize the features of interest.
Therefore, the main advantages of Neurolucida include:
1.  Neurolucida is a commercial product that is under active support and development to ensure that the product works as intended and that the results are properly analyzed and presented.
2.  Neurolucida controls the microscope hardware allowing users to trace off a live image or efficiently acquire images.  ImageJ typically works on already acquired images; while Filament Tracer does not control microscopic hardware and does not offer manual tracing and reconstruction from brightfield image stacks.
3.  Neurolucida analyzes images, calculates and presents results for users, while ImageJ allows users to write their own plug-ins.
4.  Neurolucida support staff offers dedicated software and hardware support allowing users focus on their research.  This support is not provided for users of ImageJ.

Comparison of Stereo Investigator system with competitors:

  1. New CAST:  Olympus New Cast stereology program is difficult to use and requires extensive knowledge of stereology making it unsuitable for a core facility.  The program is not designed for multi-channel fluorescent stereology. While it includes methodology for optical fractionator probes, it does not include similar support for Space Balls (for length estimation) or Nearest Neighbor analysis (for clustering). 
  2. Bioquant:  The stereology program from Bioquant (Bioquant, Nashville, TN) is even more limited than New Cast.  It has limited stereological capabilities and no probes for Space Balls or Nearest Neighbor analysis.  It is not designed for multi-channel fluorescence and does not control the Zeiss M1 microscope.

 

The Stereo Investigator system combines accuracy, speed and superb image quality with the modularity that is essential to address the needs of these diverse levels of analysis in a single system.  The requirements of the major users in this proposal fall into three categories:

  1. Stereology using multiple fluorescent labels;
  2. Archival collection of large image montages;
  3. Neuron tracing and quantitative reconstruction.

For a single system to satisfy all three requirements, the microscope system must be precisely configured.  The proposed MBF Bioscience system is unique in its ability to satisfy each of these; utilizing an integrated control interface to reduce the training burden, while increasing throughput and efficiency of the costly investment. The proposed system is expandable and adaptable in accordance with the changing needs and profile of the major user group.
Current-generation software systems available do not demonstrate the integration between hardware components and software control of the proposed MBF system. Table 2 compares the most powerful computational and graphics computer systems currently available on the market today, with the performance of the proposed system (provided by MBF Bioscience, March 16, 2009).

Table 1:  Comparison of Available Stereology Programs

 

Stereo Investigator

New CAST

Bioquant

Work with multi-channel fluorescent images

Yes

Yes

No

Work with confocal or brightfield image stacks

Yes

Limited

No

Create virtual slides

Yes

No

No

Off-line data collection/analysis

Yes

No

No

Optical Fractionator Workflow

Yes

No

No

Add comments inside a probe for later analysis

Yes

No

No

Automatically calculate results

Yes

No

Yes

Automatically calculate CE estimates

Yes

No

Limited

Nearest Neighbor Analysis

Yes

No

No

Space Balls Probe

Yes

No

No

Direct Data export to Microsoft Excel

Yes

No

No